Microbial fermentation processes are applied for industrial production of a broad and rapidly expanding range of chemical compounds from renewable carbohydrate feedstocks.
Especially in anaerobic fermentation processes, redox balancing of the cofactor couple NADH/NAD+ can cause important constraints on product yields. This challenge is exemplified by the formation of glycerol as major by-product in the industrial production of—for instance—fuel ethanol by Saccharomyces cerevisiae, a direct consequence of the need to reoxidize NADH formed in biosynthetic reactions.
Ethanol production by Saccharomyces cerevisiae is currently, by volume, the single largest fermentation process in industrial biotechnology, but various other compounds, including other alcohols, carboxylic acids, isoprenoids, amino acids etc, are currently produced in industrial biotechnological processes.
Various approaches have been proposed to improve the fermentative properties of organisms used in industrial biotechnology by genetic modification.
WO 2008/028019 relates to a method for forming fermentation products utilizing a microorganism having at least one heterologous gene sequence, the method comprising the steps of converting at least one carbohydrate to 3-phosphoglycerate and fixing carbon dioxide, wherein at least one of said steps is catalyzed by at least one exogenous enzyme. Further, it relates to a microorganism for forming fermentation products through fermentation of at least one sugar, the microorganism comprising at least one heterologous gene sequence encoding at least one enzyme selected from the group consisting of phosphopentose epimerase, phosphoribulokinase, and ribulose bisphosphate carboxylase.
In an example, a yeast is mentioned wherein a heterologous PRK and a heterologous Rubisco gene are incorporated. In an embodiment the yeast is used for ethanol production. The results (FIG. 24) show concentrations for transgenic controls and the modified strains. Little difference is noticeable between modified yeast and its corresponding control. No information is apparent regarding product yield, sugar conversion, yeast growth, evaporation rates of ethanol. Thus, it is apparent that results are not conclusive with respect to an improvement in ethanol yield.
Further, WO 2008/028019 is silent on the problem of glycerol side-product formation.
A major challenge relating to the stoichiometry of yeast-based production of ethanol, but also of other compounds, is that substantial amounts of NADH-dependent side-products (in particular glycerol) are generally formed as a by-product, especially under anaerobic and oxygen-limited conditions or under conditions where respiration is otherwise constrained or absent. It has been estimated that, in typical industrial ethanol processes, up to about 4 wt. % of the sugar feedstock is converted into glycerol (Nissen et al. Yeast 16 (2000) 463-474). Under conditions that are ideal for anaerobic growth, the conversion into glycerol may even be higher, up to about 10%.
Glycerol production under anaerobic conditions is primarily linked to redox metabolism. During anaerobic growth of S. cerevisiae, sugar dissimilation occurs via alcoholic fermentation. In this process, the NADH formed in the glycolytic glyceraldehyde-3-phosphate dehydrogenase reaction is reoxidized by converting acetaldehyde, formed by decarboxylation of pyruvate to ethanol via NAD+-dependent alcohol dehydrogenase. The fixed stoichiometry of this redox-neutral dissimilatory pathway causes problems when a net reduction of NAD+ to NADH occurs elsewhere in metabolism. Under anaerobic conditions, NADH reoxidation in S. cerevisiae is strictly dependent on reduction of sugar to glycerol. Glycerol formation is initiated by reduction of the glycolytic intermediate dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate (glycerol-3P), a reaction catalyzed by NAD+-dependent glycerol 3-phosphate dehydrogenase. Subsequently, the glycerol 3-phosphate formed in this reaction is hydrolysed by glycerol-3-phosphatase to yield glycerol and inorganic phosphate. Consequently, glycerol is a major by-product during anaerobic production of ethanol by S. cerevisiae, which is undesired as it reduces overall conversion of sugar to ethanol. Further, the presence of glycerol in effluents of ethanol production plants may impose costs for waste-water treatment.
In WO 2011/010923, the NADH-related side-product (glycerol) formation in a process for the production of ethanol from a carbohydrate containing feedstock—in particular a carbohydrate feedstock derived from lignocellulosic biomass—glycerol side-production problem is addressed by providing a recombinant yeast cell comprising one or more recombinant nucleic acid sequences encoding an NAD+-dependent acetylating acetaldehyde dehydrogenase (EC 1.2.1.10) activity, said cell either lacking enzymatic activity needed for the NADH-dependent glycerol synthesis or the cell having a reduced enzymatic activity with respect to the NADH-dependent glycerol synthesis compared to its corresponding wild-type yeast cell. A cell is described that is effective in essentially eliminating glycerol production. Also, the cell uses acetate to reoxidise NADH, whereby ethanol yield can be increased if an acetate-containing feedstock is used.
Although the described process in WO 2011/010923 is advantageous, there is a continuing need for alternatives, in particular alternatives that also allow the production of a useful organic compound, such as ethanol, without needing acetate or other organic electron acceptor molecules in order to eliminate or at least reduce NADH-dependent side-product synthesis. It would in particular be desirable to provide a microorganism wherein NADH-dependent side-product synthesis is reduced and which allows increased product yield, also in the absence of acetate.
The inventors realised that it may be possible to reduce or even eliminate NADH-dependent side-product synthesis by functionally expressing a recombinant enzyme in a heterotrophic, chemotrophic microorganism cell, in particular a yeast cell, using carbon dioxide as a substrate.